The signaling adapter proteins called TNF receptor associated factors (TRAFs) play important roles in mediating and regulating the functions of receptors of both adaptive and innate immunity. As such, they are attractive potential targets for small molecule drug design and biological therapeutics. However, the nature of their roles in signaling can be quite context-dependent, so it is imperative to fully understand how they interact with both other TRAFs and with receptors in regulating signaling and ultimate downstream cellular functions. The goals of the proposed project are to fill several important remaining knowledge gaps in this area, with a continuation of the previous project period[unreadable]s overall focus on TRAF function in lymphocytes. The first Aim will characterize how TRAF3 interacts with TRAF5 and TRAF6 to regulate signaling to lymphocytes by members of both the TNFR and TLR receptor families. This Aim will build upon new data demonstrating TRAF3-TRAF5 and TRAF3-TRAF6 associations in cells that have the potential to exert important regulatory functions both at the cell membrane and in the cytoplasm. The second Aim continues the theme of investigation of TRAF-TRAF and TRAF-receptor interactions, investigating the molecular mechanisms by which TNFR superfamily members BAFFR and TACI utilize TRAF molecules in B cells, and how these associations regulate signaling by these receptors. Experiments in Aim 2 will characterize TRAF interactions with BAFFR and TACI in B lymphocytes following interaction with BAFF and APRIL, determine which TRAF molecules are functionally required for BAFFR and TACI signaling, and determine the role of the TRAF and receptor-interacting protein Act1 in mediating CD40, BAFFR and TACI signaling.